title: Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges.
abstract: S100A3, a member of the EF-hand-type Ca(2+)-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn(2+) affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca(2+)/Zn(2+) supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca(2+) binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca(2+) affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 Å resolution shows that SS1 renders the C-terminal classical Ca(2+)-binding loop flexible, which are essential for its Ca(2+) binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn(2+) by (Cys)(3)His residues that is compatible with SS2 formation in S100A3.
authors.affiliation: Frontier Research Center for Applied Atomic Sciences, Ibaraki University, 162-1 Shirakata, Tokai, Naka, Ibaraki 319-1106, Japan. unno19@mx.ibaraki.ac.jp
authors.forename: Masaki
authors.initials: M
authors.lastname: Unno
authors.forename: Takumi
authors.initials: T
authors.lastname: Kawasaki
authors.forename: Hidenari
authors.initials: H
authors.lastname: Takahara
authors.forename: Claus W
authors.initials: CW
authors.lastname: Heizmann
authors.forename: Kenji
authors.initials: K
authors.lastname: Kizawa
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D018360:Crystallography, X-Ray; D004220:Disulfides; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D009418:S100 Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D004220:Disulfides; D050505:Mutant Proteins; D009418:S100 Proteins; C082356:S100A3 protein, human; D015032:Zinc; D002118:Calcium